Liu Yali, Zeng Chao, Bao Nandi, Zhao Jie, Hu Yuzhen, Li Chengxin, Chi Sumin
Department of Physiology, State Key Discipline of Cell Biology, The Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China.
Team 2, Cadet Brigade, School of Stomatology, Chinese People's Liberation Army General Hospital, Beijing 100853, P.R. China.
Oncol Rep. 2015 Oct;34(4):1835-44. doi: 10.3892/or.2015.4152. Epub 2015 Jul 24.
Rab23, as a negative regulatory molecule of the Hedgehog (Hh) signaling pathway, may be a new target for treating carcinoma. In the present study, we aimed to determine whether Rab23 is expressed in breast cancer cells and whether Rab23 affects the viability and proliferation of breast cancer cells. We evaluated Rab23 expression in several breast cancer cell lines including MDA-MB-231, Bcap37 and MCF-7 by reverse transcription-PCR (RT-PCR), western blotting and immunofluorescence in vitro. We assessed cell growth and proliferation by 3-(4,5-dimethylthiazol‑2-y1)‑3,5-diphenyltetrazolium bromide (MTT), colony formation and bromodeoxyuridine (BrdU) incorporation assays. The distribution of the cell cycle and the rate of apoptosis were assessed using flow cytometry (FCM). In addition, we determined the mechanisms by which Rab23 regulates the Hh pathway by detecting the level of Gli molecules by RT-PCR. We found that Rab23 mRNA and protein levels were expressed in breast cancer cells, and the expression of Rab23 in MDA-MB-231 cells was higher than that in the MCF-7 cells. Rab23 protein was primarily expressed and localized in the cytoplasm surrounding the nucleus. The MTT assay showed that the absorbance value at A(490 nm) of the Rab23‑transfected group was reduced in comparison with the control group. The number of colonies formed in the breast cancer cells was significantly reduced and BrdU labeling was weakened in the group transfected with Rab23. The results of FCM showed that overexpression of Rab23 protein caused cell cycle arrest in the G1 phase and a decrease in the S phase population as well as induction of apoptosis. Furthermore, Rab23 decreased Gli1 and Gli2 mRNA levels when compared with the control group. Our results indicate that Rab23 is expressed in breast cancer cells, and ectopic expression of Rab23 inhibits the growth and proliferation as well as induces cell apoptosis in breast cancer cells. These effects may be due to the inhibition by Rab23 of Gli1 and Gli2 mRNA expression. These results suggest that Rab23 is a potential target for the treatment of breast cancer.
Rab23作为刺猬信号通路（Hh）的负调控分子，可能成为治疗癌症的新靶点。在本研究中，我们旨在确定Rab23是否在乳腺癌细胞中表达，以及Rab23是否影响乳腺癌细胞的活力和增殖。我们通过逆转录聚合酶链反应（RT-PCR）、蛋白质免疫印迹法和体外免疫荧光技术，评估了Rab23在包括MDA-MB-231、Bcap37和MCF-7在内的几种乳腺癌细胞系中的表达。我们通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）、集落形成和溴脱氧尿苷（BrdU）掺入试验评估细胞生长和增殖。使用流式细胞术（FCM）评估细胞周期分布和凋亡率。此外，我们通过RT-PCR检测Gli分子水平，确定Rab23调节Hh信号通路的机制。我们发现Rab23 mRNA和蛋白在乳腺癌细胞中表达，且MDA-MB-231细胞中Rab23的表达高于MCF-7细胞。Rab23蛋白主要表达并定位于细胞核周围的细胞质中。MTT试验表明，与对照组相比，Rab23转染组在A(490 nm)处的吸光度值降低。在转染Rab23的组中，乳腺癌细胞形成的集落数量显著减少，BrdU标记减弱。FCM结果表明，Rab23蛋白的过表达导致细胞周期停滞在G1期，S期细胞群体减少以及诱导细胞凋亡。此外，与对照组相比，Rab23降低了Gli1和Gli2 mRNA水平。我们的结果表明，Rab23在乳腺癌细胞中表达，Rab23的异位表达抑制乳腺癌细胞的生长和增殖并诱导细胞凋亡。这些作用可能是由于Rab23对Gli1和Gli2 mRNA表达的抑制。这些结果表明，Rab23是治疗乳腺癌的潜在靶点。
